Bulletin of Entomological Research (2013) 103, 451–457 doi:10.1017/S0007485313000035 © Cambridge University Press 2013 Lysiphlebus orientalis (Hymenoptera, Braconidae), a new invasive aphid parasitoid in Europe – evidence from molecular markers Andjeljko Petrovic´1*, Milana Mitrovic´2, Petr Starý3, Olivera Petrovic´-Obradovic´4, Vladimir Žikic´5, Željko Tomanovic´1 and Christoph Vorburger6 1Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia: 2Department of Plant Pests, Institute for Plant Protection and Environment, Banatska 33, Zemun, Serbia: 3Laboratory of Aphidology, Institute of Entomology, Biology Centre, Academy of Sciences ˇ of the Czech Republic, Branišovská 31, 37005 Ceské Budeˇjovice, Czech Republic: 4Department of Plant Protection, Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11081 Zemun, Serbia: 5Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš,Višegradska 33, 18000 Niš, Serbia: 6Institute of Integrative Biology, ETH Zürich, Switzerland, and EAWAG, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, 8600 Dübendorf, Switzerland Abstract We report the occurrence of Lysiphlebus orientalis in Serbia, an aphid parasitoid from the Far East that is new to Europe and has the potential to become invasive. Our finding based on morphological characters is confirmed by analyses of mitochondrial cytochrome oxidase subunit I sequences. An increase in number and an expansion of the host range were observed during field studies over the past two years, and it is determined that the current host range encompasses nine aphid hosts on 12 different host plants, forming 13 tri-trophic associations. A host range determined for European populations of L. orientalis appears wider compared with that in its Far Eastern native habitats where Aphis glycines Mats. is the sole known host. Moreover, it overlaps considerably with the host ranges of European parasitoids that play an important role in the natural control of pest aphids. Keywords: new invasive species, Serbia, DNA barcoding, biological control, parasitoid (Accepted 17 January 2013; First published online 1 March 2013) Introduction & Schwalbe, 2002). While invasions of herbivorous pest arthropods are typically the consequence of unintentional Non-native arthropods abound worldwide and many introductions, predators and parasitoids of pest arthropods cause substantial ecological and economic damage (Hallman are often introduced deliberately for biological control. Nevertheless, there are numerous cases of alien arthropod predators and parasitoids that became invasive and have *Author for correspondence undesirable effects on the invaded ecosystems (Roy et al., Phone: +381 11 2638 890 2011). Here we report the case of an accidentally introduced Fax: +381 11 2638 500 parasitoid of aphids in Europe that appears to be expanding E-mail: [email protected] its host range. Downloaded from https://www.cambridge.org/core. Library of Eawag, Empa, PSI & WSL, on 11 Sep 2019 at 12:32:17, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007485313000035 452 A. Petrovic´ et al. The subfamily Aphidiinae (Hymenoptera, Braconidae) research on Aphidiinae fauna. Plant samples infested with consists of solitary endoparasitoids of aphids, several of live and mummified aphids were collected in the field and which are of great value in pest aphid control (Hagvar & transferred into plastic containers covered with nylon mesh. Hofsvang, 1991, Brewer & Elliott, 2004) and are commercially Voucher specimens of live aphids from each plant sample available as biocontol agents (Boivin et al., 2012). More than were preserved in 70% ethanol for later identification. The 200 species have been recorded in Europe (van Achterberg, remaining aphids were maintained in the laboratory until 2011), but there is variation in how comprehensively the fauna parasitoid emergence. of Aphidiinae is documented in different areas/countries. The external morphology of emerged parasitoids was With 100 recorded species, Serbia is one of the most studied using a ZEISS Discovery V8 stereomicroscope. All extensively surveyed European countries (Petrovic´ et al., materials used in this study are deposited in the collection 2009, Petrovic´ et al., 2011). The genus Lysiphlebus Förster, of the Institute of Zoology, Faculty of Biology, University of with about 30 known species, belongs to a taxonomically Belgrade. and biologically less studied genera (Mackauer, 1961; Kambhampati et al., 2000). It comprises highly specialized species such as L. hirticornis Mackauer which parasitize DNA extraction, polymerase chain reaction (PCR) amplification Metopeurum fuscoviride exclusively (Nyabuga et al., 2009), as and sequencing well as relative generalists such as a L. testaceipes (Cr.) with a Thirty-five Lysiphlebus specimens were used for the mol- host range of more than 50 species in Europe (Starý et al., 2004) ecular confirmation of species status and for the analysis of and even more in its native home, North America (Pike et al., genetic variability (table 1). L. orientalis was represented with 2000). An important characteristic of the genus Lysiphlebus 22 specimens originating from Serbia and five from China. We is the occurrence of species that have sexual and asexual also obtained sequences from two specimens of L. testaceipes reproduction such as L. fabarum, L. confusus and L. cardui and three specimens of L. balcanicus. Both species are members (summarized in Sandrock et al., 2011). of the ‘testaceipes’ group which is closely related to L. orientalis. Aphidiinae parasitoids have been used in biological control In addition to members of the ‘testaceipes’ group, we programs against pest aphids on various crops worldwide sequenced three specimens of L. fabarum which shares aphid (Hagvar & Hofsvang, 1991). Although new biocontrol agents hosts with L. orientalis. Prior to DNA extraction, all specimens are typically subject to a risk assessment prior to their field were stored in 96% ethanol at À20°C. DNA was extracted from release, post-release studies often focus on establishment each individual adult wasp using the KAPA Express Extract success and target effects, paying less attention to the effects kit (Kapa Biosystems) following the manufacturer’s instruc- of introduced parasitoids on non-target hosts. However, the tions. A region of approximately 710bp of the barcoding best example on how important the latter studies are is region of the mitochondrial cytochrome oxidase subunit I L. testaceipes, a biocontrol agent introduced to Mediterranean (COI) gene was amplified using the primers LCO1490 (5′- Europe from Cuba to control Aphis citricola and Toxoptera GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 aurantii in citrus crops. Within less than three decades (5′-TAAACTTCAGGCTGACCAAAAAATCA-3′) (Folmer L. testaceipes expanded its host range to 50 different aphid et al., 1994). species (Cecilio, 1994; Starý et al., 1988, 2004), replacing native DNA amplification was performed in a final volume of parasitoid species. In 2008, L. testaceipes was removed from the 25μl containing 1μl of the extracted DNA, 1×KAPA2G Robust positive list of biocontrol agents in Europe by the European HotStart ReadyMix (contains 2mM MgCl at 1X) and 0.5μM Plant Protection Organization (EPPO 2008-03-26/28). 2 of each primer. All PCRs were conducted in an Eppendorf Lysiphlebus orientalis Starý & Rakhshani was recently Mastercycler® using the following thermal profile: initial described from northeast China as a specialized parasitoid denaturation at 95°C for 5min, followed by 35 cycles of 94°C of the soybean aphid (Aphis glycines Matsumura) on Glycine for 60s, 54°C for 60s, 72°C for 90s and a final extension step max (L.). On the basis of field-collected material and laboratory at 72°C for 7min. The PCR products were purified using rearing, Starý et al.(2010) determined that this parasitoid the QIAquick PCR Purification Kit (Qiagen) according to the occurs in all-female populations that reproduce by thelyto- manufacturer’s instructions, while DNA sequencing in both kous parthenogenesis. Since the soybean aphid has become directions was performed by Macrogen Inc. (Seoul, Korea). a major source of economic loss for soybean production in North America (Heimpel et al., 2010; Ragsdale et al., 2011), L. orientalis is currently under investigation as a potential Genetic analysis biocontrol agent (Starý et al., 2010; Ragsdale et al., 2011). In their description of L. orientalis, Starý et al.(2010) determined Sequences were edited using FinchTV (www.geospiza. on the basis of morphological characters that some Lysiphlebus com). After multiple alignments, conducted using CLUSTAL specimens found recently in Europe may be related to W integrated in MEGA5 software (Tamura et al., 2011), L. orientalis. Here we present molecular evidence that sequences showed no indels (insertion/deletion) and were L. orientalis is indeed present in Europe, presumably due to trimmed to a length of 630bp. All sequences were deposited an accidental introduction. It was collected repeatedly in under accession numbers KC237734-KC237768 in GenBank. Serbia and it appears to be spreading geographically as well as For calculation of average genetic distances between se- expanding its host range. quences, within each group and between groups of species, we used Kimura’s two-parameter method (K2P) of base